PROJECT SUMMARY Invariant natural killer T cells (iNKT cells) are a fascinating, innate-like T lymphocyte population that recognizes glycolipids presented by CD1d, a class I-like antigen- presenting molecule. Mice deficient for iNKT cells are highly susceptible to infection by inhalation with Streptococcus pneumoniae (S. pneumoniae), a pathogen that is responsible for many deaths worldwide. We have identified glycolipid antigens from S. pneumoniae that activate the T cell antigen receptor (TCR) of iNKT cells, and we have shown TCR-dependent activation of these cells to produce IFN or IL-17 within hours of infection. While it is established that iNKT cells augment the innate immune response in the lung, there is little information as to how they do this, and if the separate iNKT cell subsets dedicated to IFN or IL-17 production behave differently. In aim one, we will use imaging and other technologies to track the iNKT cell response in the lung of infected mice. We will identify the cell types the subsets of iNKT cells interact with, their recruitment and expansion in situ, and their dynamic behavior. In aim two, we will determine how the innate immune response of myeloid cells and epithelial cells to S. pneumoniae is affected by the absence of iNKT cells, or iNKT cell production of IFN and/or IL-17. We have shown that the Ig super family member BTLA is important for epithelial innate responses by binding to the TNF super family receptor HVEM. Therefore, also in Aim 2, we will determine if BTLA expression by iNKT cells is required, which would be suggestive of a novel, direct interaction of iNKT cells with epithelial cells. Alternatively, a requirement for BTLA expression by another cell type, combined with data from our intravital imaging studies, would be suggestive of an indirect iNKT- epithelial cell interaction. In aim three, we will identify the cell type(s) that must express CD1d for a protective response to infection using mice with a floxed Cd1d allele. Our hypothesis is that iNKT cells activate diverse elements of innate immunity in the lung, including epithelial cells, and that IFN and IL-17 producing iNKT cells have non redundant roles in host defense. The results from these studies will provide novel information on the innate immune response in the lung and how iNKT cells modulate this response. The increased understanding of iNKT cell function we will gain may contribute to the improved design of glycolipid adjuvants that stimulate iNKT cells to combat infections.